Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant

Pseudomonas aeruginosa is the second-leading cause of nosocomial infections and pneumonia in hospitals. Because of its extraordinary capacity for developing resistance to antibiotics, treating infections by Pseudomonas is becoming a challenge, lengthening hospital stays, and increasing medical costs and mortality. The outer membrane protein OprF is a well-conserved and immunogenic porin playing an important role in quorum sensing and in biofilm formation. Here, we used a bacterial cell-free expression system to reconstitute OprF under its native forms in liposomes and we demonstrated that the resulting OprF proteoliposomes can be used as a fully functional recombinant vaccine against P. aeruginosa. Remarkably, we showed that our system promotes the folding of OprF into its active open oligomerized state as well as the formation of mega-pores. Our approach thus represents an easy and efficient way for producing bacterial membrane antigens exposing native epitopes for vaccine purposes.

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Faculty Opinions recommendation of MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.12481.469965 ◽

2006 ◽

Author(s):

Michio Hirano

Keyword(s):

Mitochondrial Dna ◽

Membrane Protein ◽

Mitochondrial Membrane ◽

Inner Mitochondrial Membrane ◽

Mitochondrial Dna Depletion ◽

Mitochondrial Membrane Protein

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Mechanism of estrogen receptor-dependent transcription in a cell-free system

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6607-6612.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6607-6612

Author(s):

J F Elliston ◽

S E Fawell ◽

L Klein-Hitpass ◽

S Y Tsai ◽

M J Tsai ◽

...

Keyword(s):

Estrogen Receptor ◽

Rna Polymerase Ii ◽

Expression System ◽

Steroid Receptor ◽

Free Expression ◽

Independent Manner ◽

Free System ◽

A Cell ◽

Regulated Gene Expression ◽

Cell Free Expression System

RNA synthesis was stimulated directly in a cell-free expression system by crude preparations of recombinant mouse estrogen receptor (ER). Receptor-stimulated transcription required the presence of estrogen response elements (EREs) in the test template and could be specifically inhibited by addition of competitor oligonucleotides containing EREs. Moreover, polyclonal antibodies directed against the DNA-binding region of ER inhibited ER-dependent transcription. In our cell-free expression system, hormone-free ER induced transcription in a hormone-independent manner. Evidence is presented suggesting that ER acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II. These observations lend support to our current understanding of the mechanism of steroid receptor-regulated gene expression and suggest strong conservation of function among members of the steroid receptor superfamily.

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Abstract 437: Optimizing a Prokaryotic Cell Free Expression System for the Generation of synthetic ApoB-Containing Lipoprotein Particles

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a437 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Humra Athar ◽

Zhenghui G Jiang ◽

Christopher J McKnight

Keyword(s):

Density Gradient ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Expression System ◽

Low Density Lipoproteins ◽

Free Expression ◽

Low Density ◽

Prokaryotic Cell ◽

Lipoprotein Particles ◽

Cell Free Expression System

High serum levels of low density lipoproteins (LDL) is associated with increased risk of atherosclerosis. Apolipoprotein B (ApoB) is required for the assembly and secretion of chylomicrons and very low density lipoproteins (VLDL), the precursor of low density lipoproteins (LDL). Despite its clinical significance, the mechanism of the assembly of these ApoB containing lipoproteins is poorly understood. The assembly process is an interplay of several key components including but not limited to nascent ApoB, lipids, ER resident chaperones and importantly, microsomal triglyceride transfer protein (MTP). In the current study, we are trying to understand several unanswered questions in the mechanism of the lipoprotein assembly. We have used a novel prokaryotic cell-free expression system and lipids mimicking the ER membrane to produce particles that represent the early dense initiation particles formed in the ER. After optimizing several different conditions, we were able to make “synthetic” lipoproteins by cotranslational expression of constructs from the first 22% of ApoB tagged with a 6-histidine tag at the C-terminus (ApoB 22-His) with small unilamellar phosphatidylcholine (PC) vesicles and phosphatidylcholine:triolein (PC:TO) emulsions. After cotranslational interaction with lipids, these constructs migrate to a lower density in potassium bromide (KBr) density gradient centrifugation. Here we report a new ApoB 22 construct with a FLAG tag at the N-terminus in addition to the C-terminal His tag. The construct makes significant amount of soluble protein that is soluble in the cell free reaction. The two N- and C-terminal tags allow us to purify full length construct from any truncation products. In addition, the dual-tag approach will allow us to purify the synthetic lipoproteins directly from the cell free system, and thereby avoid the requirement for KBr density gradient centrifugation. This new strategy will provide far more efficient generation and purification of synthetic ApoB containing lipoprotein particles.

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Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

Clinical and Vaccine Immunology ◽

10.1128/cvi.00496-09 ◽

2010 ◽

Vol 17 (5) ◽

pp. 784-792 ◽

Cited By ~ 34

Author(s):

R. Zichel ◽

A. Mimran ◽

A. Keren ◽

A. Barnea ◽

I. Steinberger-Levy ◽

...

Keyword(s):

Expression System ◽

Free Expression ◽

Enzyme Linked Immunosorbent Assay ◽

High Dose ◽

Botulinum Toxins ◽

Free System ◽

Toxin Complex ◽

A Cell ◽

Cell Free Expression System

ABSTRACT Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni+ affinity chromatography. Mice immunized with three injections containing 5 μg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 105 against the native toxin complex, which enabled protection against a high-dose toxin challenge (103 to 106 mouse 50% lethal dose [MsLD50]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 105 MsLD50 toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

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